This application is a 371 of PCT/FR00/00093 Jan. 18, 2000.
The present invention relates to phosphinic pseudo-peptides that may particularly be used as very powerful and specific inhibitors for matrix zinc metalloproteases MMP, particularly MMP-11, MMP-2, MMP-9 and MMP-8. However, these pseudo-peptides appear to show a low activity with respect to MMP-1 and MMP-7. In this way, these inhibitors offer the possibility to only inhibit one MMP sub-family in vivo, and could therefore prove to be less toxic than MMP inhibitors with a very broad spectrum of activity.
Said pseudo-peptides may have applications in the treatment of diseases characterised by the over-expression of matrix proteases, such as connective and joint tissue degeneration, rheumatoid arthritis, osteoarthritis, aortic aneurysm and cancer.
On the basis of the studies conducted in vivo on animals, these phosphinic inhibitors appear to allow use in pharmaceutical formulations to inhibit primary or secondary tumour growth.
Matrix zinc metalloproteases MMP represent a family of enzymes collectively capable of splitting all the proteins of the extracellular matrix. Due to their role on extracellular matrix proteins, these enzymes play a very important role during the development and the course of various tissue remodelling processes, such as involution of the mammary gland, cicatrisation and extravasion of specialised immune response cells.
Around fifteen enzymes in humans belonging to the MMP family are known to date:
Collagenases appear to be the only MMPs to be capable of splitting collagen in fibrillary form. Gelatinases A and B are characterised by their ability to split type IV collagen, which is very abundant in the basal membranes and collagen in denatured form. Stromelysins 1 and 2 appear to be responsible for the breakdown of other proteins of the extracellular matrix, such as fibronectin and various proteoglycans. MT-MMPs appear to be above all involved in the activation of gelatinase A, and due to their membrane location, these matrixins appear to play a role as a membrane receptor of gelatinase A. It is important to note that the physiological substrate of stromelysin-3 is not known to date. However, several studies on this protease, which was initially characterised in breast tumours, suggest that stromelysin-3 is an important factor in the development and survival of tumours.
MMPs appear to be over-expressed in various human diseases, particularly cancer. In this disease, for a long time, the role of MMPs was associated with the invasion of tumour cells and their ability to pass through various barriers to form secondary tumours. More recently, various studies have established that said proteases certainly play a more fundamental role in carcinogenesis, particularly by taking part in primary tumour growth. Of different theories to explain this function of MMPs, the most studied relate to their role in:
their ability, by means of extracellular matrix protein proteolysis, to release from said matrix the growth factors essential for the development and survival of tumours, and
angiogenesis, required for tumour growth.
The apparent involvement of MMPs in tumour growth has led numerous teams world-wide to focus on the role of compounds capable of inhibiting these enzymes.
Synthesis programmes on MMP inhibitors were initiated a number of years ago. At this time, applications of MMP inhibitors particularly related to inflammatory diseases of the connective tissue. It was only more recently that multiple programmes on the application of MMP inhibitors in cancerology have developed, as described by Brown, Medical Oncology, 1997, 14, 1-10, [1]. Indeed, as mentioned above, the studies demonstrating that MMPs must be considered as priority targets in the development of new anticancer agents are relatively recent. As such, it may be noted that, in 1997, 67 patents relating to MMP inhibitors were registered world-wide, the majority of which relate to cancerology applications, as described by Beckett et al, 1998, Exp. Opin. Ther. Patents, 8, p.259-289 [2]. In most of these patents, the synthesised compounds belong to the family of pseudo-peptide derivatives comprising a hydroxamate function. Some patents relate to pseudo-peptide compounds comprising carboxyl-alkyl or thiol functions. In said compounds, the hydroxamate, thiol or carboxyl-alkyl functions interact with the zinc atom present in the active MMP site. The most advanced compounds in terms of anticancer activity have been developed by the firm British Biotechnology. Two compounds, Batimastat BB94 and Marimastat have been the subject of phase II and III clinical studies in humans. Since then, other firms (Roche, Bayer, Agouron, Novartis) appear to have conducted phase I and II clinical studies on MMP inhibitors in cancerology. 
In this way, none of the compounds liable to inhibit MMPs known by means of references [1] and [2], are composed of a phosphinic pseudo-peptide.
However, the document: Goulet et al, Bioorg. Med. Chem. Lett. 4, 1994, p. 1221-1224 [3], describes phosphinic pseudo-peptides that can be used as a selective inhibitor of stromelysin-1 (MMP-3), which comprise the final sequence: 
Caldwell et al, Bioorg. Med. Chem. Letter 6, 1996, p.323-328 [4], also describe a phosphinic pseudo-peptide which is a selective inhibitor of stromelysin-1 (MMP-3), which comprises the same terminal sequence as the pseudo-peptide of reference [3].
In these references [3] and [4], it is attempted to detect activity with respect to MMP-3, while in the invention, it is attempted to detect a selective activity with respect to MMP-11, MMP-2, MMP-9 and MMP-8. It is also important to note that, in the present invention, R3 is not only a phenylethyl residue. In addition, the invention demonstrates that other substituents in this position give compounds with increased inhibitory power.
The documents FR-A-2 676 059 [5], FR-A-2 689 764 [6] and EP-A-0 725 075(7] illustrate phosphinic pseudo-peptides showing an inhibitory activity with respect to bacterial collagenases and zinc endopeptidases 24.15 and 24.16.
The present invention specifically relates to new phosphinic pseudo-peptides showing a powerful and specific inhibitory activity with respect to the matrix zinc metalloproteases MMP-11, MMP-2, MMP-9 and MMP-8.
According to the invention, the phosphinic pseudo-peptide complies with the formula: 
wherein
R1 is a group inhibiting an amine function, or an amino acid residue or peptide with an amino-terminal group protecting function,
R2 represents the lateral chain of a natural or non-natural amino acid,
R3 represents:
1) the lateral chain of a natural amino acid except for Gly and Ala, not substituted or substituted by an aryl group,
2) an aralkyl group, or
3) an alkyl group comprising at least 3 carbon atoms, and
R4 represents a lateral chain of natural or non-natural amino acid, or a dinitrobenzyl group.
Said pseudo-peptide according to formula I is a pseudo-tripeptide comprising a phosphinic type chemical group, the function of which is to chelate the zinc atom in MMPs. In said pseudo-tripeptide, the choice of the R2, R3 and R4 groups makes it possible to ensure the interaction of the tripeptide with the MMP sub-sites S1, S1xe2x80x2 and S2xe2x80x2, respectively. The R1 group is assumed to interact at the junction of the sub-sites S3/S2.
In the above definition of the pseudo-peptides according to the invention, the terms xe2x80x9camino acidxe2x80x9d used for R1, R2, R3 and R4 in [5] refer to the twenty xcex1-amino acids commonly found in proteins which are also known as standard amino acids and their analogues. The lateral chains of said amino acids comprise linear and ramified alkyl, hydroxyalkyl, carboxyalkyl, aralkyl, aminoalkyl, carboxamide alkyl, mercapto alkyl, phenylalkyl, hydroxyphenylalkyl, guanidinoalkyl, imidazoylalkyl, indolylalkyl, and pyrrolidinyl groups.
Examples of amino acid that may be used include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, iso-leucine, leucine, norleucine, lysin, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine, nitrophenylalanine, homo-arginine, thiazolidine and dehydroproline.
However, in the case of R3, the amino acid cannot be Gly or Ala since they do not show sufficient interaction with the MMP sub-site S1xe2x80x2.
Preferentially, the amino acid used for R3 is chosen from Phe, Leu or Ser, Cys residues wherein the lateral chain is substituted by an arylalkyl group.
The aryl groups liable to be used are those derived from a monocyclic or polycyclic aromatic core, possibly substituted by alkyl or alcoxy groups. Examples of aryl groups that may be used include the phenyl, naphthyl, benzyl and alcoxybenzyl groups, such as p-methoxybenzyl.
R3 may also represent an aralkyl group. In said aralkyl group, the aryl group may be any of the other mentioned above. The alkyl part of the aralkyl group may be a linear or ramified chain of 1 to 6 carbon atoms.
Examples of the aralkyl groups that may be used include groups according to the formulas: 
where n is an integer from 1 to 4, and 
where p is equal to 1 or 2.
The alkyl groups that may be used for R3 have at least 3 carbon atoms. They may be linear or ramified and preferentially have at most 7 carbon atoms. Examples of alkyl groups that may be used include the groups CH3xe2x80x94(CH2)6xe2x80x94 and (CH3)2xe2x80x94CHxe2x80x94CH2xe2x80x94.
Preferentially, R3 represents a group complying of any of the following formulas: 
According to the invention, R2 is chosen so as to interact with the MMP sub-site S1. Good results are obtained when R2 represents the methyl or benzyl group; preferentially, R2 is the benzyl group, which corresponds to the lateral chain of Phe.
According to the invention, R4 is chosen so as to interact with the MMP sub-site S2xe2x80x2. Good results are obtained when R4 represents the lateral chain of Trp or a dinitrobenzyl group Dpa.
In the pseudo-peptide according to the invention, the lateral chains R2, R3 and R4 of the amino acids may be in L or D form. In addition, the pseudo-peptide may be composed of a single isomer or by a mixture of 4-diasteroisomers due to the presence of two asymmetrical centres on the xcex1 carbon comprising the R2 and R3 residues. Although any amino acid configuration may be suitable, it is preferable that the unit: 
has an L configuration.
However, three out of four diastereoisomers corresponding to different R2 and R3 configurations have practically equivalent activities as MMP inhibitors.
In the pseudo-peptides according to the invention, R1 may represent various groups, the nature of which influences the affinity of the pseudo-peptide with respect to the different MMPs.
R1 may represent a xe2x80x9cgroup inhibiting an amine functionxe2x80x9d. These terms include all the inhibiting groups that may be used to inhibit the amine functions of amino acids and peptides, for example t-butoxy-carbonyl, benzyloxycarbonyl, cinnamoyl, pivalolyl and N-(I-fluorenyl-methoxycarbonyl) Fmoc groups.
R1 may also represent inhibiting groups chosen from the acetyl, benzyloxyacetyl, phenylaminoacetyl, (m-chlorophenyl)aminoacetyl, (2-hydroxy-5-chloro-phenyl) amino acetyl, indolyl-2-carbonyl, 4,6-dichloro-indolyl-2-carbonyl, quinolyl-2-carbonyl and 1-oxa-2,4-dichloro-7-naphthalene carbonyl groups, or an amino acid or peptide residue wherein the terminal amine function is inhibited by a suitable group. Examples of such residues include the Z-Ala and Z-Leu groups wherein Z represents the benzyloxycarbonyl group.
The pseudo-peptides according to the invention may be prepared using conventional methods from phosphinic blocks according to the formula: 
wherein Z represents the benzyloxycarbonyl group and Ad the adamantyl group, and the amino acid corresponding to R4 by solid phase chemical synthesis according to the methods described by Yotakis et al, J. Org. Chem., 1996, 61, page 6601-6605 [8] and Jiracek et al, J. Biol. Chem., 1995, 270, p. 21701-21706 [9] and J. Biol. Chem., 1996, 271, p. 19606-19611 [10].
The initial phosphinic blocks may be obtained by Michael addition of a phosphinic acid comprising the R2 group 
on an acrylate supplying the R3 group 
where Et represents the ethyl group.
The acrylates supplying the R3 group may be synthesised using different processes, as seen below.
The pseudo-peptides according to the invention may be used in the treatment of diseases involving an over-expression of matrix zinc metalloproteases.
The invention also relates to a pharmaceutical formulation inhibiting at least one matrix zinc metalloprotease, comprising at least one pseudo-peptide according to formula I as defined above.
Preferentially, said formulation inhibits a matrix zinc metalloprotease chosen from MMP-2, MMP-8, MMP-9 and MMP-11.
Said formulation is intended to treat a disease characterised by the over-expression of matrix proteases, such as cancer.
The invention also relates to the use of a phosphinic pseudo-peptide according to formula I as defined above, to manufacture a medicinal product inhibiting at least one matrix zinc metalloprotease, particularly MMP-2, MMP-8, MMP-9 and MMP-11.
The invention""s other characteristics and benefits will be seen more clearly upon reading the following description, naturally given as a non-restrictive illustration, in relation to the appended figures.
FIG. 1 is a synthesis diagram of pseudo-peptides according to the invention.
FIG. 2 is a diagram illustrating the anti-tumoral efficacy of a compound according to the invention, which gives the mean volume of the tumour (in mm3) as a function of time (in days) after injecting cancerous cells in control mice and mice treated with the pseudo-peptide according to the invention.